Vegetative compatibility and parasexual
segregation in Colletotrichum lindemuthianum,
a fungal pathogen of the common bean
M.A.A. Castro-Prado, C.B. Querol, J.R. Sant’Anna, C.T. Miyamoto,
C.C.S. Franco, C.A. Mangolin and M.F.P.S. Machado
Departamento de Biologia Celular e Genética, Universidade Estadual de
Maringá, Maringá, PR, Brasil
Corresponding author: M.A.A. Castro-Prado
E-mail: [email protected]
Genet. Mol. Res. 6 (3): 634-642 (2007)
Received May 18, 2007
Accepted August 2, 2007
Published September 30, 2007
ABSTRACT. The heterokaryotic and vegetative diploid phases of
Colletotrichum lindemuthianum are described using nutritional and
biochemical markers. Nitrate non-utilizing mutants (nit), derived
from R2047, R89, R73, R65, and R23 isolates, were paired in all
possible combinations to obtain heterokaryons. Although pairings
R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete
vegetative incompatibility, prototrophic heterokaryons were obtained
from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89,
R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23.
Heterokaryons gave rise to spontaneous mitotic segregants which
carried markers corresponding to one or the other of the parental strains.
Heterokaryons spontaneously produced prototrophic fast-growing
sectors too, characterized as diploid segregants. Diploids would be
expected to yield auxotrophic segregants following haploidization
in basal medium or in the presence of benomyl. Parental haploid
segregants were in fact recovered from diploid colonies growing in
basal medium and basal medium containing the haploidizing agent.
Although barriers to the formation of heterokaryons in some crosses
Genetics and Molecular Research 6 (3): 634-642 (2007)
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were detected, the results demonstrate the occurrence of parasexuality
among vegetative compatible mutants of C. lindemuthianum.
Key words: Colletotrichum lindemuthianum, Heterokaryosis,
Esterase isoenzymes, Nitrate non-utilizing mutants,
Vegetative compatibility groups
INTRODUCTION
Colletotrichum lindemuthianum (teleomorph Glomerella lindemuthiana) is the causal agent
of anthracnose, one of the main diseases of the common bean (Phaseolus vulgaris). Anthracnose
occurs principally in leaf veins and pods, with almost total production loss (Jerba et al., 2005).
Although the sexual phase of C. lindemuthianum has already been reported in the laboratory, sexual
reproduction in field conditions is not common. However, the pathogen shows high genetic variability
which interferes with the cultivars’ resistance durability, with subsequent difficulty in the effective
control of anthracnose (Kimati and Galli, 1970; Balardin et al., 1997; Mahuku and Riascos, 2004).
The formation of conidial anastomosis tubes between conidia within acervuli has been
observed during C. lindemuthianum conidiogenesis and described as a mechanism that favors
exchange of nuclear material and organelles between incompatible strains. Conidial anastomosis
tubes were detected in pairs or in multiple associations of conidia (Roca et al., 2003).
Hyphal anastomoses among vegetative compatible isolates also favor exchange of
genetic material in filamentous fungi and lead to the formation of vegetative heterokaryons
(i.e., cells containing two genetically different nuclei), which have been characterized as
the first stage of the parasexual cycle (Pontecorvo et al., 1953; Saupe, 2000). Haploid
nuclei in the heterokaryotic mycelium fuse together and form diploid nuclei. Through
successive processes of chromosome non-disjunction, the latter give rise to paternal
haploids or recombinants (Pontecorvo et al., 1953; Miyamoto et al., 2007).
The viability of heterokaryons is regulated by multiple vegetative incompatibility loci, named
vic (for vegetative incompatibility) or het (for heterokaryon incompatibility), so that only compatible
strains, believed to be clonally related, may produce stable heterokaryons among themselves (Saupe,
2000). Actually, nitrate non-utilizing mutants (nit) of Magnaphorte grisea and Fusarium species have
been used to force heterokaryons between nearly isogenic strains, and to test isolates for vegetative
compatibility (Klittich and Leslie, 1988; Baayen et al., 1998; Correl et al., 2000).
The aim of the present study was to obtain nit mutants from C. lindemuthianum isolates
and to use these auxotrophic mutants to obtain heterokaryons and parasexual segregants. Whereas
nitrate non-utilizing mutants were used to form heterokaryons and diploids, native polyacrylamide
gel electrophoresis was used to show the differential expression of α- and β-esterase in haploid
and diploid strains of C. lindemuthianum isolates, as an additional marker.
MATERIAL AND METHODS
Strains
Races R2047 and R23 of C. lindemuthianum were kindly donated by Dr. Maria Celeste
Vidigal (Departamento de Agronomia, Universidade Estadual de Maringá, Paraná, Brazil), while
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M.A.A. Castro-Prado et al.
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races R89, R73 and R65 were obtained from the Embrapa Arroz e Feijão, Goiás, Brazil. Culture
stocks were maintained at 5°C in amber flasks with basal medium (BM).
Media
BM was prepared with 1000 mL distilled H2O, 30 g saccharose, 1 g KH2PO4, 0.5 g MgSO4ּ 7
H2O, 0.5 g KCl, 10 mg FeSO4ּ 7 H2O, 15 g agar, and 0.2 mL trace element solution (95 mL distilled
H2O, 5 g citric acid, 1 g Fe(NH4)2(SO4)2ּ 6 H2O, 0.25 g CuSO4ּ 5 H2O, 50 mg MnSO4ּ H2O, 50 mg
H3BO3, and 50 mg Na2MoO4ּ 2 H2O). Minimal medium (MM) consisted of Czapek-Dox with 1%
(w/v) glucose and 15 g agar. Complete medium (CM) consisted of 1000 mL distilled H2O, 10 g
glucose, 2 g peptone, 0.5 g yeast extract, 1 g hydrolyzed casein, 4 mg inositol, 2 mg choline chloride,
2 mg pantothenic acid, 1 mg nicotinic acid, 1 mg riboflavin, 0.1 mg 4-aminobenzoic acid, 0.5 mg folic
acid, 0.5 mg pyridoxine, 0.2 mg thiamine, and 2 µg biotin added to MM.
Isolation of mutants
Mutants that were unable to use nitrate as a nitrogen source (nit) were obtained in BM
+ NaNO3 (0.2%) + KClO3 (3.0%). Mycelium plugs 5 mm in diameter were removed from the
periphery of C. lindemuthianum colonies cultivated in CM and transferred to the center of petri
dishes with BM + NaNO3 + KClO3. Dishes were incubated at 22°C for 25 days. Chlorate-resistant
mutants were then identified, isolated and transferred to petri dishes with BM, supplemented with
different nitrogen sources: BM + sodium nitrate (0.2%), BM + potassium nitrite (0.085%), BM +
hypoxanthine (0.01%), and BM + ammonium tartrate (0.092%). Plates were incubated at 22°C for
12 days, and mutants were phenotypically characterized according to Klittich and Leslie (1988).
Hyphal anastomoses
Complementary nit mutants from the isolates were paired in all possible combinations to
determine their capacity for anastomosis. Mycelium plugs of isolates were placed 0.5 cm from each
other on sterilized microscope slides covered with a thin layer of BM + NaNO3. Slides were then
placed on petri dishes containing the same medium. The plates were incubated at 22°C for 8 days.
Afterwards, the mycelium plugs were removed from the slides; the newly grown hyphae were
stained with cotton blue in lactophenol, coverslipped and examined under a light microscope.
Obtaining heterokaryons and diploids
Mycelium plugs (5 mm) of each nit mutant were paired equidistantly apart (approximately 1.0
cm) on petri dishes containing BM + NaNO3 for vegetative complementation tests. Dishes were incubated
at 22°C for 12 to 21 days and then examined for prototrophic heterokaryotic growth. Prototrophic (nit+)
and homogeneous fast-growing sectors arising from heterokaryons grown in BM + NaNO3 were isolated
and transferred to BM + NaNO3 + benomyl (0.5 µg/mL) to detect the presence of unstable diploids.
Isolation of parasexual segregants
Two methods were used to detect parasexual segregants (or sectors): 1) Isolation of
sectors showing nit phenotype, directly from the heterokaryotic or prototrophic colonies.
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Mycelium plugs (5 mm) of these sectors were transferred to BM supplemented with different
nitrogen sources for phenotypic characterization. 2) Suspensions of conidia were obtained from
heterokaryons and diploid colonies growing in BM + NaNO3. Aliquots of these suspensions were
later grown in BM + NaNO3 to recover recombinants and paternal segregants. Segregant ploidy
was determined in BM + NaNO3 + benomyl (0.5 µg/mL): segregants that failed to produce
mitotic sectors in the presence of the haploidization agent were classified as haploid segregants.
Electrophoretic analysis
Mycelium plugs of nit mutants from R2047 and R23 races were individually grown
in liquid CM. Cultures were kept at 22°C, under shaking conditions, for five days. Mycelia
obtained were blotted on filter paper after filtration and harvested individually. Two hundred
milligrams mycelium from each strain was separately homogenized with a glass rod in an
Eppendorf microcentrifuge tube using 80 µL 1.0 M phosphate buffer, pH 7.0, containing 5%
PVP-40, 1.0 mM EDTA, 0.5% β-mercaptoethanol, 0.5% triton X-100, and 10% glycerol
solution. After homogenization, the samples were centrifuged at 14,000 rpm for 30 min at
4°C in a Beckman GS-15R centrifuge, and the supernatant (35 µL) was used from each
sample. The polyacrylamide gels (15%) were prepared with 0.375 M Tris-HCl, pH 8.8, as
buffer (Ceron et al., 1992). Electrophoresis was performed for 8 h at 4°C, at a constant voltage
of 200 V. Running buffer was 0.1 M Tris-glycine, pH 8.3. Staining techniques following a
protocol established for plants (Pereira et al., 2001) identified the esterases. The gels were
soaked for 30 min in 50 mL 0.1 M sodium phosphate, pH 6.2, at room temperature. Esterase
activity was visualized by placing the gels for 1 h in a staining solution prepared from 50 mL
sodium phosphate solution, 40 mg β-naphthyl acetate, 40 mg α-naphthyl acetate, 60 mg Fast
Blue RR salt, and 5.0 mL N-propanol. Polyacrylamide gels were dried as described by Ceron
et al. (1992) and Lapenta et al. (1995), and kept at room temperature for 1 h in a mixture
of 7.5% acetic acid and 10% glycerol; the gels were then embedded in 5% gelatin, placed
between two sheets of wet cellophane paper stretched on an embroidering hoop and left to
dry for 24-48 h. The α-preferential esterases hydrolyzed preferentially α-naphthyl acetate in
the presence of α- and β-naphthyl acetate and formed black bands; the β-preferential esterases
hydrolyzed preferentially β-naphthyl acetate in the presence of α- and β-naphthyl acetate and
formed red bands, and α/β-esterases hydrolyzed equally α- and β-naphthyl acetate, forming
a mixture of black and red bands. The α-, β- and α/β-esterases were numbered in sequence,
starting from the anode, according to decreasing negative charge.
Results
Isolation of nitrate non-utilizing mutants
Stable mutants obtained in the presence of potassium chlorate, which produced
poor growth colonies, or rather, without dense aerial mycelium, were considered mutants
incapable of using nitrate (nit). Mutants were screened for their ability to utilize different
nitrogen sources in order to determine their phenotypes and could be divided into three distinct
phenotypic classes: nit1 (nitrate non-utilizing), Nit3 (nitrite and nitrate non-utilizing), and NitM
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(hypoxanthine and nitrate non-utilizing). One hundred and twenty-eight nitrate non-utilizing
mutants were obtained from the five C. lindemuthianum isolates. Seventy-six (59%) were
classified as nit1 mutants, nineteen (15%) were characterized as Nit3 mutants, and thirty-three
(26%) typified NitM mutants, utilizing nitrite but not hypoxanthine (results not shown). The
remaining mutants, which showed either phenotypic instability when grown in BM + NaNO3
or could utilize nitrate, were discarded. These unstable mutants were characterized as either
heterokaryons containing both nit and nit+ nuclei, or containing a distinct mutation conferring
both nitrate-utilizing and chlorate-resistance phenotypes.
Anastomoses between isolates and heterokaryon formation
All isolates were tested for anastomosis and heterokaryon formation. Isolates R2047,
R65 and R23 were found to carry out anastomosis among themselves but none of them formed
anastomoses with the R73 isolate. R89 isolate, on the other hand, was capable of carrying out
anastomosis with all the isolate ones, except with R2047 (Figure 1A).
Figure 1. A. Hyphal anastomoses (arrows) among nit mutants of R2047 and R23 isolates. B. Pairing between NitM
mutants from R65 and R23 races separated by a dialysis membrane. Heterokaryons (arrows) did not occur beneath the
dialysis membrane which impairs direct contact between the strains’ mycelia. C. Heterokaryotic colony showing mitotic
segregation of the nit paternal mutants (arrows). D. Heterokaryons (arrow) formed between NitM mutants of R65 (1, 3,
4), R23 (2), and R2047 (5). E. Heterokaryon colony showing a fast-growing sector (arrow). F. Unstable diploid strain
growing in BM + benomyl.
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Pairing of complementary nit mutants produced colonies of prototrophic growth in
BM + NaNO3, after two or three weeks of incubation at 22°C. Since no heterokaryotic growth
occurred when the paired mutant strains were separated by a dialysis membrane (Figure
1B), this fact indicated that the prototrophic growth is not due to cross-feeding. Prototrophic
colonies gave rise spontaneously to auxotrophic sectors (or parasexual segregants) in BM +
NaNO3, which showed parental (i.e., nit1, Nit3 or NitM) phenotypes (Table 1, Figure 1C).
Results indicate that the prototrophic growth from the pairing of different complementary
auxotrophic mutants must be due to heterokaryosis.
To determine whether the isolates were vegetatively self-compatible, nit1 mutants were
paired with either Nit3 or NitM mutants from the same strain on BM + NaNO3. Mutants were
seen to be self-compatible due to the formation of dense heterokaryons (results not shown).
When pairing the complementary nit mutants of R2047, R65 and R23 isolates in all possible
combinations, interstrain barriers of vegetative incompatibility were not observed. Therefore,
isolates R2047, R23 and R65 were allocated in the vegetative compatibility group I (VCG I).
In addition, mutants NitM of R2047, R65 and R23 races showed vegetative complementation
among themselves and formed a dense line of growth in the contact area between the colonies
(Table 1, Figure 1D). On the other hand, pairings R2047/R89, R2047/R73, R73/R65, and R73/
R23 showed complete vegetative incompatibility by this method (results not shown), and R73
isolate was allocated in another vegetative compatibility group (VCG II).
Diploid formation and characterization
Fast-growing sectors arise spontaneously from prototrophic heterokaryotic colonies
two or three weeks after pairing in BM + NaNO3 (Figure 1E). Sectors showed nit+ phenotype
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M.A.A. Castro-Prado et al.
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and growth rate similar to that of the original wild-type strains in BM + NaNO3. When conidia
or mycelium plugs from prototrophic sectors were transferred to BM + NaNO3 or benomylsupplemented BM + NaNO3, their mitotic instability could be identified by the production of
auxotrophic segregants (or sectors), which showed parental (NitM or nit1) phenotypes (Table 1,
Figure 1F). The results suggest that fast-growing sectors are at least transiently diploid.
The diploid strains R2047.4 (NitM) // R23.16 (NitM) and R2047.4 (NitM) // R23.19
(NitM), and the parental haploid strains, R2047.4, R23.16 and R23.19, were characterized
for esterase isoenzyme expression, and differential α- and β-esterase patterns were observed
(Figure 2). A total of 27 α- and β-esterases were identified, but EST-7, EST-13, EST-17,
and EST-22 were present only in the R2047.4 haploid strain. EST-6, EST-8, EST-14, EST16, EST-18, EST-20, EST-23, EST-24, EST-25, and EST-26 isoenzymes were detected only
in the R23.16 and R23.19 strains. Strain-specific esterases indicate specific biochemical
characteristics, and differential expression of esterase loci have in fact been demonstrated in
the filamentous fungus Aspergillus nidulans (Machado and de Castro-Prado, 2001). On the
other hand, in diploid strain R2047.4 // R23.19, both paternal and additional (EST-9, EST-10,
EST-12, EST-14, and EST-27) isoenzyme esterases were detected (sample 3, Figure 2).
Figure 2. Polyacrylamide gel electrophoresis showing α- and β-esterases in haploid strains R2047.4 (sample 1), R23.19
(sample 2), and R23.16 (sample 4), and in diploid strains R2047.4 (NitM) // R23.19 (NitM) (sample 3), and R2047.4
(NitM) // R23.16 (NitM) (sample 5). The Est-7, Est-13, Est-17, and Est-22 isozymes were present only in the R2047.4
haploid strain, whereas Est-6, Est-8, Est-14, Est-16, Est-18, Est-20, Est-23, Est-24, Est-25, and Est-26
isozymes were detected only in the R23.16 and R23.19 strains.
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DISCUSSION
Nitrate non-utilizing mutants were used to demonstrate heterokaryosis and parasexual
segregation in C. lindemuthianum isolates. Auxotrophic complementation and segregation of
nutritional markers have also been used to characterize parasexuality in M. grisea and F.
moniliforme (Crawford et al., 1986; Klittich and Leslie, 1988).
Heterokaryon formation by the anastomosis of homokaryotic hyphae contributes to the
high degree of genetic variability encountered in filamentous fungi, and provides a mechanism
for the restoration of normal function in combined, separately deficient, genomes (Zeigler,
1998; Saupe, 2000). Contrastingly, somatic incompatibility, which impairs heterokaryon
formation among different mycelia, may affect the parasexual occurrence (Saupe, 2000). In the
present study, races R2047, R65 and R23 were shown to integrate the VCG I, whereas the R73
isolate was allocated in VCG II. Vegetative incompatibility between R2047 and R73 isolates
were also reported by Roca et al. (2004). On the other hand, nit mutants from R89 isolate were
able to carry out anastomosis and to form heterokaryons with nit mutants of isolates from both
VCG I (R65 and R23) and VCG II (R73). The R89 isolate may be characterized as a “bridge”
strain, analogous to the C. lindemuthianum Mi.7.99 isolate (Rodríguez-Guerra et al., 2003),
which was capable of carrying out anastomosis with isolates from two anastomosis groups.
In Neurospora crassa and Fusarium species, multiple loci control the production of
a molybdenum-containing co-factor (Tomsett and Garrett, 1980; Klittich and Leslie, 1988).
Prototrophic recombinants were in fact recovered from meiotic crosses of nitM mutants of
F. moniliforme strains of opposite mating type (Klittich and Leslie, 1988). The vegetative
complementation of nit mutants with the same phenotype shown in this study (Figure 1D, Table
1), also suggests the existence of multiple loci that control the production of the molybdenumcontaining co-factor in C. lindemuthianum. Further studies will be necessary to determine how
many loci are involved in the production of the NitM phenotypes.
The association of genetically dissimilar nuclei in heterokaryotic mycelia may form
stable heterozygous diploid nuclei through the fusion of two complete genomes. Diploid
colonies can be distinguished easily from haploids on the basis of their restrictive growth
and breakdown in the presence of benomyl, the haploidization agent. Benomyl may produce
haploidy via successive chromosome losses and mitotic non-disjunction (Pontecorvo et al.,
1953; Hastie, 1970; Miyamoto et al., 2007). In the present study, diploid strains were obtained
spontaneously from heterokaryons formed between complementary nit mutants. The results
of haploidization of the diploid strains, presented in Table 1, demonstrate that heterokaryons
in C. lindemuthianum may yield unstable diploid conidia. Although all diploid strains gave
rise to haploid paternal segregants, the phenotypic analyses of these segregants demonstrated
that certain parental genotypes were not recovered or recovered only at low frequency. The
differential expression of esterase loci in diploid and respective haploid paternal strains is more
an evidence of the occurrence of parasexuality in the C. lindemuthianum pathogen, underpinning
the fact that different forms of nucleosome organization in chromatin may increase or repress
the expression of specific genes (Lee and Young, 2000), or that recombination events in diploid
strains may generate differential α- and β-esterase phenotypes.
The present study provides evidence of both heterokaryosis and diploid formation in
C. lindemuthianum under laboratory conditions, using nitrate non-utilizing mutants.
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